Array assays between surface bound binding agents or probes and target molecules in solution are used to detect the presence of particular biopolymers in a sample. The surface-bound probes may be oligonucleotides, peptides, polypeptides, proteins, antibodies or other molecules capable of binding with target molecules of interest in solution. Such binding interactions are the basis for many of the methods and devices used in a variety of different fields, e.g., genomics (in sequencing by hybridization, single nucleotide polymerase (SNP) detection, differential gene expression analysis, identification of novel genes, gene mapping, finger printing, etc.) and proteomics.
One typical array assay method involves biopolymeric probes immobilized in an array on a substrate. A solution containing analytes that bind with the attached probes is placed in contact with the array substrate, covered with another substrate such as a coverslip or the like to form an assay area and placed in an environmentally controlled chamber such as an incubator or the like. Usually, the targets in the solution bind to the complementary probes on the substrate to form a binding complex. The binding by target molecules to biopolymer probe features or spots on the substrate produces a pattern on the surface of the substrate and provides desired information about the sample. In most instances, the target molecules are labeled with a detectable tag such as a fluorescent tag or chemiluminescent tag.
The resultant binding interaction or complexes of binding pairs are then detected and read or interrogated, for example by optical means, although other methods may also be used. Typically, imaging devices or analogous instruments designed to map the density of sample adhered to a biopolymer array are employed to read an array. For example, laser light may be used to excite fluorescent tags, generating a signal only in those spots on the biochip that have a target molecule and thus a fluorescent tag bound to a probe molecule. This pattern may then be digitally scanned for computer analysis.
As such, optical scanners play an important role in many array-based applications. Optical scanners act like a large field fluorescence microscope in which the fluorescent pattern caused by binding of labeled molecules on the array surface is scanned. In this way, a laser induced fluorescence scanner provides for analyzing large numbers of different target molecules of interest, e.g., genes/mutations/alleles, in a biological sample.
The scanning equipment typically used for the evaluation of arrays includes a scanning fluorometer. A number of different types of such devices are commercially available from different sources, such as Perkin-Elmer, Agilent, or Axon Instruments., etc. Analysis of the data, (i.e., collection, reconstruction of image, comparison and interpretation of data) is performed with associated computer systems and commercially available software, such as Quantarray™ by Perkin-Elmer, Genepix Pro™ by Axon Instructions, Microarray Suite™ by Affymetrix, as well as Feature Extraction Software and Rosetta Resolver Gene Expression Data Analysis System, both available from Agilent.
In such devices, a light source (e.g., a laser light source) generates an excitation light, e.g., a collimated beam. The excitation light is focused on the array and sequentially illuminates small surface regions of known location on an array substrate. The resulting fluorescence signals from the surface regions are collected employing the same lens used to focus the laser light onto the array, or off-axis (using a separate lens positioned to one side of the lens used to focus the laser onto the array). The collected signals are then typically transmitted through appropriate spectral filters, to an optical detector. A recording device, such as a computer memory, records the detected signals and builds up a raster scan file of intensities as a function of position, or time as it relates to the position. Such intensities, as a function of position, are typically referred to in the art as “pixels”. In obtaining array data, an important parameter is the scale factor of the scanner. The scale factor is defined as the number of signal counts that are reported to the user per chromophore per area on the array, e.g., counts in a 10 micron pixel/chromophore per square micron. Different chromophores or dyes employed in array-based applications typically have different scale factors for a given detection system. For a scanner this would mean that different dyes would have a different scale factors even for equivalent detector gain and intensity of excitation light. This variation in the scale factor for different dyes depends a given dye's quantum efficiency, extinction coefficient, excitation and emission spectra.
Typically, the scale factor employed by a scanner during use is programmed or set for a specific dye or dyes during manufacturing of the scanner. This programming or setting is done by fixing the detector gain (i.e. voltage for a photo-multiplier tube (PMT) detector) or by varying the intensity of the excitation light incident on the array. For example, where a scanner is designed to read an array in two different channels, the scanner is typically programmed to employ a given gain setting to obtain a first scale factor in the first channel and another gain setting to obtain a second scale factor in the second channel, where the programmed scale factors are those that are appropriate for specific first and second dyes, e.g., Cy3 and Cy5. Such preprogramming usually ensures that the same scale factor is employed for each of the two supported dyes each time an array is scanned. Obtaining equal scale factors for different dyes generally requires different gain settings for each dye for the reasons described above. In some cases different scale factors can intentionally be set for different dyes. Problems with such preprogrammed scanners may arise where a user employs a scanner with dyes other than the dyes for which the scanner was initially programmed. For example, in scanners that are set or programmed for use with Cy3 and Cy5, other markers can be employed in such scanners, but the readings obtained therefrom will be readings that are taken with Cy3 and Cy5 scale factors. The scale factors for the specific dyes employed may be different and may not be appropriate. As such, errors in terms of actual intensity and its relation to the density of targets may be introduced, thereby skewing the obtained results. Further, if the scale factor for a specific dye is different enough it may lead to signals saturating the detector or falling below the detection limit of the scanner.
Currently, where users wish to employ a scanner with dyes other than those for which the scanner is designed, the user must make manual changes to the detector gain (i.e. change PMT voltage) to maintain constant scale factor. Alternatively, users can normalize obtained data after scanning to remove errors introduced by the incorrect scale factor. This post-scan normalization step may not be possible if the scan is saturated or below the detection limit due to a substantially different scale factor
While the above approaches may remove many of the errors introduced by incorrect scale factor usage, they require additional steps. As such, there is interest in the development of scanners that can be used with a plurality of different dyes, where the scanner is capable of automatically selecting a correct scale factor to use for a given dye during the scan. The present invention satisfies this, and other, needs.
Relevant Literature
United States Patents of interest include: U.S. Pat. No. 5,091,652; 5,260,578; 5,296,700; 5,324,633; 5,585,639; 5,760,951; 5,763,870; 6,084,991; 6,222,664; 6,284,465; 6,317,370 6,320,196 and 6,355,934. See also Li et al., Abstract 27. Fully Automated Multiplexed Capillary Systems for DNA Sample Analysis, DOE Human Genome Program, Contractor-Grantee Workshop VIII (Feb. 27 to Mar. 2, 2000) Santa Fe, N.M.